Despite improvements in the management of oral squamous cell carcinoma (SCC), the 5-year survival rate has remained disappointingly low, at about 40%, for the past several decades. Therefore, this indicates the need for new strategies to improve outcome. One such strategy is to better understand the earliest forms of oral SCC, in the precancerous stage, with the long-term aim of preventing the progression to fully developed disease. Oral epithelial dysplasia is the single most important risk factor for oral cancer; however, only about 20% of all patients with epithelial dysplasia will ever progress to malignancy. Presently, for the individual patient with dysplasia, no reliable biomarkers have been discovered that indicate increased risk of progression to oral SCC. Our long-term goal is to identify molecular biomarkers that will predict oral SCC development. The objectives of this proposed research are to determine the mRNA expression levels of specific novel biomarkers, identified in our microarray studies of oral SCC, in routinely processed biopsies of oral precancers and to determine if these changes are associated with the progression to oral cancer. The central hypothesis is that analysis of gene expression of specific biomarkers can be used to distinguish which oral premalignancies will progress to cancer from those which will not. The rationale for the proposed research is that by studying oral precancers, where it is known which progressed to oral SCC, critical genetic events necessary for oral cancer development can be established. This phased application will be significant because it will provide new molecular insights into the development of oral cancer and it will identify potential new targets for screening and therapeutic intervention. In the R21 phase, we will establish a reliable, quantitative PCR method for gene expression in routinely processed oral biopsies. Specific Aim 1: To optimize quantitative real-time PCR for the analysis of gene expression in laser microdissected (LCM) routinely processed oral biopsies. Specific Aim 2: To establish the quantitative requirement for LCM-generated RNA for real-time PCR. In the R33 phase, we will apply this technology to conduct a large-scale, molecular epidemiological study of specific novel biomarkers, identified in our microarray studies of oral cancer, in a cohort of 96 patients with oral precancer who have been followed for several years and where it is known which patient developed oral cancer and which did not. Specific Aim 1: To determine if oral cancer-associated biomarkers are overexpressed in LCM-oral epithelial dysplasias and oral SCC using real-time PCR. Specific Aim 2: To determine if specific biomarkers can be used to predict oral cancer development in patients with oral precancer.